The modulation of the tolerance of oocytes to cryopreservation by artificial expression of water channels -vertebrates and invertebrates-

通过水通道的人工表达调节卵母细胞对冷冻保存的耐受性-脊椎动物和无脊椎动物-

基本信息

批准号：
16380190
负责人：
KASAI Magosaburo
金额：
$ 9.6万
依托单位：
Kochi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2004
资助国家：
日本
起止时间：
2004 至 2006
项目状态：
已结题

项目摘要

The permeability of the plasma membrane to water and cryoprotectants is an important factor determining the tolerance of cells to cryopreservation and suitable condition for cryopreservation. There are two pathways for the movement of water and cryoprotectants through the plasma membrane, i.e., simple diffusion across the plasma membrane and facilitated diffusion via channel processes. In this study, we tried to clarify the relationship between the permeability of the plasma membrane and cryo-sensitivity in oocytes and embryos of various species.As a model for mammals, we used mice, and the permeability to water and cryoprotectants of oocytes and embryos at various stages were examined from the viewpoint of the expression of a water/cryoprotectant channel. It was found that in embryos at the morula stage, water and some cryoprotectants moved rapidly through the plasma membrane predominantly by facilitated diffusion via aquaporin 3 (AQP3), a water/cryoprotectant channel, whereas in oocy … More tes and embryos at early cleaved stages, water and cryoprotectants moved slowly through the plasma membrane predominantly by simple diffusion.In fish and amphibians, the cryopreservation of oocytes and embryos has not been realized. We tried to find suitable conditions for the cryopreservation of the oocytes from the viewpoint of membrane permeability. We used medaka and zebrafish as models for fish, and Xenopus laevis as a model for amphibians. In medaka and zebrafish, the permeability to water and cryoprotectants of the plasma membrane was higher in immature oocytes than mature oocytes. The high permeability of immature oocytes was increased further by the artificial expression of AQP3 by injecting AQP3 cRNA into them. Similar increase in the permeability to water and cryoprotectants was observed in AQP3 cRNA-injected immature Xenopus oocytes. However, considering the large volume of the oocyte in these animals, further increase in membrane-permeability would be necessary for successful cryopreservation of oocytes.In addition, we tried to cryopreserve insect embryos, derived from parthenogenetically activated oocytes of Nasonia vitripennis. Removal of the wax layer of Nasonia embryos was effective to make them permeable to water and cryoprotectants (ethylene glycol and DMSO). When wax-removed embryos were vitrified with ethylene glycol-based solutions, many embryos survived, and some (〜30%) of them developed to larvae. Therefore, the conservation of various strains of Nasonia vitripennis by the cryopreservation of embryos would become possible. Less

质膜对水和冷冻保护剂的渗透性是确定细胞对冷冻保存的耐受性和合适的冷冻保存条件的重要因素。水和冷冻保存通过质膜运动有两种途径，即简单扩散在质膜上，并通过通道过程支持扩散。在这项研究中，我们试图澄清质膜的渗透性与各种物种的冷冻敏感性之间的关系。作为哺乳动物的模型，我们使用小鼠使用小鼠的渗透性，从含量的cryott的角度检查了各个阶段的卵母细胞和胚胎对水和胚胎的渗透性。已经发现，在莫拉阶段的胚胎中，水和一些冷冻保护剂在质膜中迅速移动，主要是通过通过含水通道蛋白3（AQP3）的扩散制备的，水/冷冻保护剂通道的扩散，而在卵子中，卵子和胚胎在早期的阶段和胚胎中通过较早的水和cry虫在卵子中的较慢，较高的雾化捕获量，并逐渐散发出较慢的雾剂，并逐渐散发出较慢的雾素，并逐渐散发出较慢的捕获剂，并散发出较慢的捕获量两栖动物，卵母细胞和胚胎的冷冻保存尚未实现。我们试图从膜渗透性的角度找到适合卵母细胞冷冻保存的条件。我们将Medaka和斑马鱼用作鱼类的模型，而Xenopus laevis作为两栖动物的模型。在Medaka和斑马鱼中，未成熟卵母细胞中对水和质膜的冷冻渗透性高于成熟的卵母细胞。通过将AQP3 CRNA注入其中，通过AQP3的人工表达进一步增加了未成熟卵母细胞的高渗透性。在AQP3 CRNA注射未成熟的异武卵母细胞中观察到了对水和冷冻塑料的渗透性的类似增加。但是，考虑到这些动物中卵母细胞的大量，对于成功的冷冻保存卵母细胞是必需的。膜 - 渗透性是必需的。此外，我们试图从鼻虫的鼻孔植物卵巢中衍生出的冷冻质昆虫胚胎。去除鼻孔胚胎的蜡层可有效地使其渗透到水和冷冻凝集剂（乙二醇和DMSO）中。当用蜡蚀的胚胎用基于乙二醇的溶液玻璃化时，许多胚胎存活了，其中一些胚胎（〜30％）发展为幼虫。因此，通过胚胎的冷冻保存来保护各种鼻虫植物。较少的